Bot. Bull. Acad. Sin. (1998) 39: 279–285

Aziz et al. — Medicinal plant, spices, mycoflora and mycotoxins

Contamination of some common medicinal plant samples and spices by fungi and their mycotoxins

Nagy Halim Aziz1,3, Youssef A. Youssef2, Moheie Z. El-Fouly1 and Lotfy A. Moussa1

1Microbiology Department, National Center for Radiation Research and Technology, P.O. Box 29, Nasr City, Post Code, 113701,Cairo, Egypt

2Microbiology Department, Faculty of Science, Ain-Shams University, Cairo, Egypt

(Received July 14, 1997; Accepted February 24, 1998)

Abstract. A total of 84 medicinal plant samples and spices were examined for the contamination of molds and mycotoxins. Ten fungal genera of different taxonomic groups were detected. Aspergillus flavus, A. parasiticus, A. niger, Fusarium oxysporum, and Penicillium viridicatum occurred most often on the medicinal plant samples. Direct determination of mycotoxins in medicinal plant samples revealed aflatoxin B1 in 17 samples at an average of from 10 to 160 ΅gkg-1, ochratoxin-A in 3 samples at an average of from 20 to 80 ΅gkg-1, and no detection of penicillic acid, zearalenone, or T-toxin. Aspergillus flavus, A. parasiticus, and A. oryzae were aflatoxin-producers, whereas, A. ochraceus, P. viridicatum, and P. variable were ochratoxin-A producers. In addition P. viridicatum, P. chrysogenum, and P. commune were penicillic acid-producers. The molds produced high concentrations of mycotoxins in synthetic medium and low to zero concentrations in the medicinal plants.

Keywords: Medicinal plants; Mycoflora; Mycotoxins; Spices; Toxigenic fungi.

Introduction

Mycotoxin contamination of various foodstuffs and agricultural commodities is a major problem in the tropics and subtropics, where climatic conditions and agricultural and storage practices are conducive to fungal growth and toxin production. Mycotoxins are secondary metabolites of mold fungi identified in many agricultural products screened for toxigenic molds (Aziz, 1987; Clevsrton and Ljunggren, 1985; Van Egmond, 1981). Aspergillus flavus, A. candidus, A. niger, A. luchuensis, A. ochraceus, A. nidulans, F. moniliforme, F. oxysporum, Alternaria alternata, Curvularia spp., Chaetomium sp., Penicillium citrinum, and Rhizopus stolonifer were reported as the most common fungi isolated from drug plants (Ayres et al., 1980; Aziz and Youssef, 1991; Misra, 1981; Roy et al., 1988; Takatori et al., 1977). The occurrence of aflatoxin in medicinal plants has already been established (Rani and Singh, 1990; Roy and Chourasia, 1990). Mycotoxins have been reported to be carcinogenic, teratogenic, tremorogenic, haemorrhagic, and dermatitic to a wide range of organisms and to cause hepatic carcinoma in man (Refai, 1988; Wary, 1981).

In Egypt, different medicinal plant samples and spices have been imported from India during the last five years. Due to the rise of an Egyptian market for these plants, the present studies have been carried out in the interests of public health and safety.

The present investigation reports the association of mycoflora with medicinal plant samples and spices, their screening for mycotoxin producing ability, and mycotoxin occurrence in plant samples under Egyptian environmental conditions.

Materials and Methods

Samples

A total of 84 samples of fourteen different medicinal plants and spices imported from India were collected from various retailers in the city of Cairo in clean labelled packets, each containing 50 g. The samples were sent to the laboratory as soon as they were collected, finely ground in a Buhler mill, and either tested on arrival or stored at 4°C to arrest any mycotoxin formation before analysis. Table 1 shows the botanical names, the part of plant used, and the number of samples examined for each medicinal plant.

Moisture Content

Medicinal plant and spice samples were dried at 60°C under vacuum for 8 to 12 h until their weight remained constant. The weight difference after drying was considered the moisture content (Aziz, 1987).

Mycological Studies

Ten grams of each sample were added to a 90 ml portion sterile 0.85% saline solution in 500 ml Erlenmeyer flasks and homogenized thoroughly on an electric shaker

3Corresponding author.


Botanical Bulletin of Academia Sinica, Vol. 39, 1998

Table 1. Description of medicinal plant samples and spices used and number of examined samples.

English name Scientific name Part of plant used No. of samples

Black cumin Nigella sativa L. Seeds 7

Fennel Foeniculum vulgare Miller Dry fruit 7

Lime tree Tilia parvifalie Ehr. Dry inflorescence 5

Absinthium Artemisia absinthium L. Whole herb & dry leaves 5

Ginger Zingiber officinale Rosoe Rhizome 5

Cinnamon Cinnamomum cassia Blume Bark or cinnamomum bark 7

Pepper mint Mentha spicata L. Leaves 7

Carob tree Ceratonia siliqua L. Dry fruits 7

Chamomile Cymbopogom schoerenthus (L.) sperg Leaves and stems 5

Saffron Crocus sativus L. Dry parts of stylles and stigma 5

Curcuma Curcuma longa L. Rhizomes 7

Worm wood Artmisia heba Succlent branches 5

Rose Rosa canina L. H Dry buds 7

Lesser galangel Alpinia officinarum Hance Rhizome 5

at constant speed for 15 min. Tenfold serial dilutions were then prepared (Aziz and Youssef, 1991). One ml portions of three suitable dilutions of the resulting medicinal plant suspension were used to inoculate Petri dishes each containing 15 ml Sabouroud’s dextrose agar containing 0.5 mg chloramphenicol/ ml medium to suppress bacterial growth. Plates were then incubated for 7–15 days at 28°C and examined visually and microscopically for the growth of molds. The isolated molds were subcultured on the above medium for identification of Aspergilli (Raper and Fennel, 1977), Penicillia (Pitt, 1979; 1985), and other molds (Domsch et al., 1981).

Screening of Aspergillus flavus and Aspergillus parasiticus Strains

Identification of presumptive A. flavus and A. parasiticus strains depends on the reserve colony colour and gross morphology of conidial heads. Isolates were tested on a new medium, Aspergillus flavus and parasiticus agar (AFPA) of Pitt et al. (1983). The production of an intense orange yellow pigment after inoculation at 30°C for 2 days, is said to be indicative of A. flavus and A. parasiticus. The presumptive production of aflatoxin by individual isolates was demonstrated using the aflatoxin production agar (APA) of Hara et al. (1974). After incubation at 27°C for 10 days, the plate cultures were examined for blue fluorescence under long wave ultraviolet light (365 nm) (UV lamp Emita-Poland).

Analysis of Mycotoxins

Extraction of mycotoxins from medicinal plant samples and spices. Samples (25 g) of ground medicinal plant samples and spices were extracted according to the method of Grabarkiewicz-Szczesna et al. (1985). In cases with large amounts of impurities and pigments, preliminary silica gel chromatography column in chloroform or in n-hexane was performed to clean up the samples.

Cultivation of molds in medicinal plant and liquid medium. Erlenmeyer flasks (250 ml) containing 50 g of

sterile mycotoxin-free medicinal plants and spices or 50 ml of yeast extract sucrose medium (2% yeast extract and 20% sucrose) were inoculated with about 106 spores per ml and incubated with shaking at 28°C for 7 days.

After incubation, the contents of each flask were mixed with 120 ml of chloroform: water (100:10, v/v) and were shaken vigorously by rotary shaker (200 rpm) overnight. The extracts were sequentially filtered through anhydrous sodium sulfate. The chloroform extracts were collected for dryness.

Thin layer chromatography (TLC) of mycotoxins. TLC was performed in all instances with pre-coated glass plates (20 Χ 20 cm of silica gel D.G.60 Merck, Darmstadt) using chloroform : acetone (9:1, v/v) and toluene : ethyl acetate : 90% formic acid (6:3:1, v/v/v) as development solvents (Golinski and Grabarkiewicz-Szczesna, 1984). The intensity of the mycotoxins was measured with a flurodensitometer CD-60 at an excitation wavelength of 365 nm and emission wavelength of 443 nm. The amount of mycotoxin extraction given is the mean of three replicates on one TLC plate, and each spot was scanned twice.

Chemical confirmation of mycotoxins. Chemical confirmation of mycotoxins was performed directly on the developed TLC plate. The plates were then sprayed with either 20% AlCl3 solution or 20% sulfuric acid and heated 10 min at 110°C and observed under 365 nm UV light. These spraying reagents were used for visualization and to increase the fluorescence intensity of the mycotoxins. The spray reagents recommended for developing or changing the colour of mycotoxin fluorescence are presented in Table 2 (Grabarkiewicz-Saczesna et al., 1985).

Results and Discussion

Fungal Contamination

The differences in fungal populations isolated from the medicinal plant samples and spices are shown in Table 3. In all cases, a total of 20 species of fungi belonging to 9


Aziz et al. — Medicinal plant, spices, mycoflora and mycotoxins

Table 2. Colour of fluorescence of mycotoxins before and after treatment with different reagents.

Toxin Colour in UV-360 nm Colour of fluorescence in UV-360 nm

without Spraying AlCl3a H2SO4b

Aflatoxin B1 Dark-blue Dark-blue Yellow-beige

Aflatoxin B2 Dark-blue Dark-blue Yellow-beige

Aflatoxin G1 Greenish-blue Greenish-blue Beige

Aflatoxin G2 Greenish-blue Greenish-blue Beige

Ochratoxin A Greenish-blue Dark-blue Greenish-blue

Penicillic acid Dark-blue Dark-blue Pink-violet

Zearalenone Gray-blue Dark-blue Gray-blue

T-2 toxin Gray-blue Dark-blue Light-greenish-blue

aAlCl3 20% solution (W/V) in ethanol.

bH2SO4 20% solution (W/V) in ethanol, sprayed plates were heated for 15 min at 110°C.

Table 3. Percentage of samples contaminated by different fungal species isolated from different medicinial plants.

% of samples yielding different species of fungi

Fungal species Black Fennel Lime Absin- Ginger Cinn- Pepper Carob Cham- Safforn Curc- Worm Rose Lesser cumin tree thium amon mint tree omile uma wood galangel

Absidia corymbifera 71 100 20 40 20 85 57 – – – 14 – 28 20

Aspergillus flavus 100 72 43 100 80 71 100 86 60 80 86 80 71 80

Aspergillus parasiticus 86 57 71 80 100 43 71 71 80 80 71 80 100 60

Aspergillus niger 57 86 100 60 80 71 86 57 60 60 71 60 86 60

Aspergillus oryzae 43 29 – 20 – 29 29 – 40 20 – – 43 80

Aspergillus ochraceus 71 57 40 – 60 57 – 43 60 – 57 40 – –

Aspergillus terreus 29 – 60 20 – 14 – 14 – 40 14 – 29 40

Aspergillus tamarii 14 29 – – 60 – 43 29 – 20 – 40 14 60

Cladosporium herbarum – 42 40 20 60 – 42 – – – – – 14 20

Penicillium viridicatum 43 71 20 – 80 – 14 – 60 – 57 60 – – Penicillium chrysogenum 14 – 60 40 40 29 – 43 40 – 43 – 57 40

Penicillium nigricans – 14 – 20 – 14 29 57 – – 43 20 14 20

Penicillium variable 86 – – 60 60 43 – – 20 40 – – 29 –

Penicillium commume – 29 20 – – 14 29 – – – 14 – – 20 Paecilomyces variotii – 57 20 20 20 – 14 – – – 43 20 57 60

Fusarium oxysporum 29 43 60 40 – – 14 – 40 20 – 40 43 –

Fusarium solani 14 – – 20 40 14 – 25 – 20 14 – – 40

Rhizopus stolonifer 85 100 20 20 20 57 71 – – – 24 – 29 20

Mucor pusillus 85 85 20 20 40 28 85 – – – 28 – – 20

Scopulariopsis brevicaulais 42 29 40 20 20 14 29 – – – 14 – 14 20

(–) No detection fungal species.

medicinal plant samples and spices were A. flavus, A. parasiticus, and A. niger. Domsch et al. (1981) postulated that the contamination of feedstuffs with fungal species was as a result of natural extraneous contamination by dust following storage in humid conditions. Fungi fall into two ecological categories: field and storage fungi. Field fungi were observed to invade developing or mature seed while it is on the plant, the major field fungi genera are: Alternaria, Helminthosporium, Fusarium, and Cladosporium. On the other hand, storage molds are those encountered on plants at moisture conditions routinely found in stored products, these fungi are principally species of Aspergillus and Penicillium.

The dominant of Aspergillus and Penicillium spp. in all examined medicinal plant samples and spices was in accord with the results of Takatori et al. (1977) and Ayres et al. (1980), who stated that Aspergillus and Penicillium spp. were the main components of cardamon, cinnamon, fennel, coriander, cumin, black cumin, and white pepper, all of which are common in the food industry. They found

genera were isolated and identified. Sixteen species were isolated from black cumin, fennel, absinthium, and lesser galangel samples with a moisture content of 6%; 15 species from lime tree cinnamon, peppermint, ginger, curcuma, and rose samples had a moisture level 4%; and 9 species were isolated from carob tree, chamomile, worm wood and saffron with a moisture content of 2.5%. In this study, the isolated species of fungi belong to the genera Absidia, Aspergillus, Cladosporium, Penicillium, Paecilomyces, Fusarium, Rhizopus, Mucor, and Scopulariopsis. The greater number of species was held to the genus Aspergillus. Seven species were recovered namely: A. flavus, A. parasiticus, A. niger, A. oryzae, A. ochraceus, A. terreus, and A. tamarii. Five species of Penicillium were isolated, namely P. viridicatum, P. chrysogenum, P. nigricans, P. variable, and P. commune. Two species were isolated from the genus Fusarium, and only one species was found in the different plant samples and spices from the other genera of fungi. The most prevalent fungi isolated from the


Botanical Bulletin of Academia Sinica, Vol. 39, 1998

a high degree of contamination in all samples. Misra (1981) and Roy et al. (1988) isolated Aspergillus flavus, A. niger, A. fumigatus, A. orchaceus, A. candidus, A. sydowi, Chaetomium dolicholrichum, F. moniliforme, Penicillium oxalicum, Alternaria, Curvularia, and Rhizopus from the seeds of Amomum subulatum, Coriandrum sativum, Cuminum cyminum, Foeniculum vulgare, Piper nigrum, Cinnamomum zeylanicum, and from the bark of Acacia catechu, all of which are commonly used drug plants.

Aspergillus niger and A. flavus or the oryzae group—especially Aspergillus flavus— were the most frequent Aspergillus species yielded in all examined medicinal plant samples in this investigation. This was in accordance with the results of Roy and Chourasia (1990), who stated that A. flavus was the main contaminant of different herbal drug samples.

Mycotoxin Production by Isolated Fungi from Medicinal Plant Samples and Spices

Table 4 shows the type of aflatoxins produced by Aspergillus spp. isolated from medicinal plant samples and spices. Aflatoxins were produced by 38 out of l09 isolates of Aspergillus in synthetic medium. Aspergillus flavus, A. parasiticus, and A. oryzae were observed to be the most common aspergilli producing aflatoxins.

Table 5 makes clear that, out of 49 strains of A. flavus, 22 produced aflatoxins in synthetic medium and only 8 produced aflatoxins in medicinal plants. Roy et al. (1988) demonstrated that A. flavus strains obtained from drug plants produced aflatoxin B1 from 0.86 to 5.24 ΅g/ml of culture filtrates. Recently Aziz and Youssef (1991) isolated A. flavus and A. parasiticus with a high tendency for aflatoxin production from some common herbal drugs and spices. The production of ochratoxin A and penicillic acid by Aspergillus and Penicillium sp. isolated from medicinal plants was recorded in Table 6. Aspergillus ochraceus and P. variable were ochratoxin A producers, whereas P. viridicatum, P. commune, and P. chrysogenum were penicillic acid producers. It was noticed that ochratoxin A and penicillic acid producing isolates were widespread on medicinal plant samples (Wyllie and Morehouse, 1977). Aspergillus ochraceus was reported to be one of the major organisms producing ochratoxin A (Aziz, 1987). Leistner and Pitt (1977) found that, out of 442 Penicillium isolates, 44 synthesized penicillic acid, 17 ochratoxin A, 11 penitrem, 10 citrinin, 6 patulin, and 3 produced both patulin and citrinin.

On the basis the present investigation, it may be concluded that the contamination of herbal drugs and spices with mycotoxins is alarming, and such products need thor

Table 4. Distribution of aflatoxin–producing fungi among medicinal plant samples and spices.

Type of fungi Total no. of Positive strains for Type of aflatoxin produced

screened fungi examined aflatoxin production B1 B2 B1 & B2 B1 & G1 B1 & G2

Aspergillus flavus 49 22 (44.90%)a 10 3 4 2 3

Aspergillus parasiticus 37 14 (37.85%)a 50 1 6 1 1

Aspergillus oryzae 15 2 (13.33%)b 2 – – – –

Aspergillus tamarii 8 0 (0 %) 0 – – – –

Total 109 38 (34.86%) 17 (15.6%) 4 (3.67%) 10 (9.17%) 3 (2.75%) 4 (3.67%)

aAflatoxins ranged from 320–750 ΅g1–1.

bAflatoxin ranged from 80–220 ΅g1–1.

Table 5. Types of aflatoxin produced by A. flavus artificially inoculated to synthetic medium and medicinal plant samples and spices.

Medicinal Number of No. of +ve isolates (average ΅gL–1) in synthetic medium No. of +ve isolates (average ΅gkg–1) in medicinal plant

plant fungal No. of +ve B1 B1 & B2 B1 & G1 B1 & G2 No. of +ve B1 B1 & B2 B1 & G1 B1 & G2 samples isolates strains strains

Black cumin 4 2 1 (4500) – – 1 (1600) 1 1 (20) – – –

Fennel 6 4 2 (1500) 2 (14400) – – 1 – – – 1 (250)

Lime tree 6 2 2 (8200) – – – 1 – 1 (150) – –

Absinthium 3 2 1 (600) – 1 (14500) – 1 1 (200) – – –

Ginger 3 1 – 1 (11200) – – 0 – – – –

Cinnamon 3 1 – – – 1 (1150) 0 – – – –

Pepermint 6 3 2 (15500) 1 (4400) – – 1 1 (80) – – –

Carob tree 3 2 1 (14800) 1 (60) – – 0 – – – –

Chamomile 3 1 1 (4950) – – – 1 1 (45) – – –

Safforn 2 0 – – – – 0 – – – –

Curcuma 2 1 – – 1 (1200) – 0 – – – –

Worm wood 0 0 – – – – 0 – – – –

Rosse 4 2 1 (440) – – 1 (3500) 1 1 (175) – – –

Lesser glange 4 1 1 (600) – – – 1 1 (100) – – –

Total 49 22 12 5 2 3 8 6 1 1




Aziz et al. — Medicinal plant, spices, mycoflora and mycotoxins

Table 6. Ochratoxin–A and penicillic acid produced by Aspergillus and Penicillium species isolated from medicinal plant samples and spices on synthetic medium.

Type of fungi screened Total number of moulds Positive strains Ochratoxin-A Penicillic acid

Aspergillus ochraceus 15 3 3a 0

Aspergillus terreus 5 0 0 0

Penicillium viridicatum 23 5 2a 3b

Penicillium chrysogenum 18 2 0 2c

Penicillium commune 3 2 0 2c

Penicillium variable 13 3 3a 0

Total 77 15 8 7

aAverage concentration (80–225 ΅gL–1).

bAverage concentration (120–185 ΅gL–1).

cAverage concentration (70–95 ΅gL–1).

Table 7. Average value (΅gkg–1) of mycotoxins present in medicinal plant samples and spices.

Medicinal plants No. of samples No. of samples Mycotoxins concentration (΅gkg–1)

examined contaminated Aflatoxin B1 Ochratoxin–A Penicillic acid Zearalenone T-2

Black cumin 5 3 30 35 – – – 20

Fennel 6 3 160 80 – – – 80

Lime tree 7 1 75 – – – –

Absinthium 7 2 25 20 – – –

Ginger 5 2 10 – – – – 10

Cinnamon 5 0 – – – – –

Pepermint 7 3 25 35 – – – – 45

Carob tree 4 1 10 – – – –

Chamomile 6 3 145 – – – – 80 – – – – 75

Safforn 5 0 – – – – –

Curcuma 3 0 – – – – –

Worm wood 4 2 90 – – – – 50

Rosse 7 0 – – – – –

Lesser glangel 5 0 – – – – –

Total 76 20 (26.3%) 17 (22.4%) 3 – – –

(–) No detection of mycotoxins under the experimental conditions.

ough inspection before being channeled to the drug and food industries.

Mycotoxin Production in Medicinal Plant Samples and Spices

As shown in Table 7, only 17 samples were found contaminated with aflatoxin B1 and three samples with ochratoxin A. Three samples of peppermint and chamomile, two samples of black cumin, fennel, ginger, and worm wood and one sample of lime tree, absinthium, and carob tree contained aflatoxin B1. The highest level was 160 ΅gkg-1 found in the fennel. Ochratoxin A was detected in one sample of black cumin, fennel, and absinthium with an average level of 35, 80, and 20 ΅gkg-1,

respectively. No mycotoxins were detected in any sample of cinnamon, saffron, curcuma, rose, or galangel. All 84 tested medicinal plants and spices were free from penicillic acid, zearalenone, and T-2 toxin. Detection limits of mycotoxins ranged from 20 to 100 ΅gkg-1. Aspergillus, Penicillium, and Fusarium are toxigenic to human beings and animals (Wyllie and Morehouse, 1977). As stated earlier, mycotoxins are produced by fungi on plants in the field before harvest or later after harvest during long storage under favourable conditions (Gedek, 1985). As these plant materials are used for preparation of traditional medicines, the possibility of aflatoxin contamination in these is implied. This is certainly a matter of great concern because humans use these medicines to treat diseases. In previous study, Rani


Botanical Bulletin of Academia Sinica, Vol. 39, 1998

and Singh (1990) found that 89% of samples of fennel, coriander, cumin, and ammi were contaminated with aflatoxin B1 at the levels 3000 ppb, 1640 ppb, 1580 ppb, and 2550 ppb, respectively. In addition, Roy et al. (1988) and Roy and Chourasia (1990) determined that the seeds of Piper nigrum and Mucuna prurians, and the barks of Acacia catechu, Coriandrum sativum, and Elettaria cardamomun were contaminated with aflatoxin B1 at levels below 20 ΅gkg-1.

Literature Cited

Ayres, G.I., T.I. Mund, and E.W. Sondin. 1980. Microbiology of Food Spices and Condiments. A Series of Books in Food and Nutrition. edn. Schmeigert, 249 pp.

Aziz, N.H. 1987. Etiology of Toxin-Producing Fungi from the Class of Deuteromycetes Occurring in Various Feed Products. Ph.D. Thesis, Agricultural University, Cracow, Poland.

Aziz, N.H. and Y.A. Youssef. 1991. Occurrenence of aflatoxins and aflatoxin-producing moulds in fresh and processed meat in Egypt. Food Addit. Contam. 3: 321–331.

Bottalico, A., P. Lerario, and A. Visconti. 1983. Production of mycotoxins (Zearalenone, Trichothecnes and moniliformin) by Fusarium species in Italy. Microbiologie-Aliments Nutr. 1: 133–142.

Clevsrton, G. and H. Ljunggren. 1985. Aflatoxin formation and the dual phenomenen. Mycopathologia 92: 129–139.

Domsch, K.H., W. Gams, and T.H. Anderson. 1981. Compendium of Soil Fungi. vol. 1 and 2, Academic Press, London.

Gedek, B. 1985. Toxins, particularly mycotoxins in feed. Deutsche. Tierδrztl. Wochenschr. 92: 215–218.

Golinski, P. and J. Grabarkiewicz-Szczesna. 1984. Chemical confirmatory tests for ochratoxin A, citrinin, penicillic acid, sterigmatocystin and Zearalenone performed directly on thin-layer chromatographic plates. J. Assoc. Off. Anal. Chem. 67: 1108–1110.

Grabarkiewicz-Szczesna, J., P. Golinski, J. Chelkowski, and K. Szebiotko. 1985. Mycotoxins in cereal grain, Part XI, Simple multidetection procedure for determination of 11 mycotoxins in cereals. Nahrung Food 3: 229–240.

Hara, S., D.I. Fennell, and C.W. Hessiltine. 1974. Aflatoxin producing strains of Aspergillus flavus detected by flurescence of agar medium under ultra-violet light. Appl. Microbiol. 27:

1118–1128.

Leistner, L. and J.I. Pitt. 1977. Miscellaneous Penicillium toxins. In J.V. Rodricks, C.V. Hesseltine, and A.M. Mehlman (eds.), Mycotoxins and Human and Animal Health. Pathotox Publ. Park Forest South Illinois, pp. 639–653.

Misra, N. 1981. Influence of temperature and relative humidity on fungal flora of some spices in storage. Z. Lebensm. Unters. Forsch. 172(1): 30–31.

Pitt, J.I. 1979. The Genus Penicillium and its Teleomorphic States. Eupenicillium and Talaromyces. Academic Press Inc. London, 634 pp.

Pitt, J.I. 1985. A laboratory guide to common Penicillium species. Commonwealth Mycological Institute, Kew, Surrey, England, 184 pp.

Pitt, J.I., D.R. Glenn, and A.D. Hocking. 1983. An improved medium for the detection of Aspergillus flavus and A. parasiticus. J. Appl. Bacteriol. 54: 109–114.

Rani, N. and S. Singh. 1990. Aflatoxin contamination of some umbelliferous spices of human use. Int. Symp. and Workshop on Food Cont. Mycotoxins and Phycotoxins, November 4-15, 1990, Cairo, Egypt, Abst. Book, pp. 79–80.

Raper, K.B. and D.I. Fennel. 1977. The “Genus Aspergillus” R.E. Krieger Publishing Company, Huntington, New York.

Refai, M.K. 1988. Aflatoxins and Aflatoxicosis. J. Egypt Vet. Med. Ass. 48(1): 1–19.

Roy, A.K. and H.K. Chourasia. 1990. Mycoflora, mycotoxin producibility and mycotoxins in traditional herbal drugs from India. J. Gen. Appl. Microbiol. 36: 295–302.

Roy, A.K., K.K. Sinha, and H.K. Chourasia. 1988. Aflatoxin contamination of some common drug plants. Appl. Environ. Microbiol. 54: 842–843.

Takatori, K., K. Watanabe, S. Udagawa, and H. Kurata. 1977. Mycoflora of imported spices and inhibitory effects of the spices on the growth of some fungi. Proc. Jpn. Assoc. Mycotoxicol. 9: 36–38.

Van Egmond, H.P. 1981. Determination of Mycotoxins. In J.F. Lawrence (ed.), Trace Analysis, vol. 1, New York, Academic Press, pp. 99–144.

Wary, B.B. 1981. Aflatoxin, hepatitis, B-virus and hepatocellular carcinoma. New England J. Med. 305(14): 833–843.

Wyllie, T.D. and L.C. Morehouse. 1977. Mycotoxic Fungi, Mycotoxins, Mycotoxicosis, Mycotoxic Fungi and Chemistry of Mycotoxins. Marcel Dekker, Inc. New York, USA.